Type I interferon signaling deficiency results in dysregulated innate immune responses to SARS‐CoV‐2 in mice

Abstract SARS‐CoV‐2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID‐19). The type I interferon (IFN) pathway is of particular importance for anti‐viral defence and recent studies identified that type I IFNs drive early inflammatory responses to SARS‐CoV‐2. Here, we use a mouse model of SARS‐CoV‐2 infection, facilitating viral entry by intranasal recombinant Adeno‐Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN‐signalling‐deficient (Ifnar1–/–) mice, to study type I IFN signalling deficiency and innate immune responses during SARS‐CoV‐2 infection. Our data show that type I IFN signaling is essential for inducing anti‐viral effector responses to SARS‐CoV‐2, control of virus replication and to prevent enhanced disease. Furthermore, hACE2‐Ifnar1–/– mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as a reduced and delayed production of monocyte‐recruiting chemokine CCL2. hACE2‐Ifnar1–/‐ mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS‐CoV‐2 infection, with a shift from Ly6C+ to Ly6C– expressing cells. Together, our findings suggest that type I IFN deficiency results in a dysregulated innate immune response to SARS‐CoV‐2 infection. This article is protected by copyright. All rights reserved

Introduction 71 The ongoing coronavirus disease  pandemic caused by severe acute respiratory syndrome-72 coronavirus 2 (SARS-CoV-2) has resulted in over 400 million cases in the first two years of the 73 pandemic. The estimated fatality rate lies between 1-2%, however this is considerably higher for 74 elderly patients over 80 years of age (~10%) and nursing home residents (>20%) (1).  (4). Furthermore, in severe and critically ill COVID-19 patients, an 83 impaired type I IFN response has been observed, resulting in decreased viral clearance (5). A lack of 84 an efficient type I IFN response in these patients is in part due to inborn errors of type I IFN immunity 85 (6) or circulating auto-antibodies neutralizing type I IFNs (7). Also, a recent animal study has identified 86 that type I IFN signaling is required for the recruitment of pro-inflammatory cells into the lungs  (9), followed by intranasal infection with 2x10 6 PFU SARS-CoV-2  Figure 1C).
140 Furthermore, Ifng gene expression was significantly increased in hACE2-Ifnar1 -/mice later during the 141 infection, by 4 d.p.i. (Supp. Figure 4A). This correlated with CD3 + T cell recruitment to the airways 142 (Supp. Figure 4B-D). Our data suggest that limited levels of type I or III IFNs are produced early during 143 infection in the Ifnar1 -/mice resulting in some ISG expression but overall, these data suggest that type 144 I IFN signaling is the main driver for inducing cell intrinsic anti-viral responses.

Introduction
The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) has resulted in over 400 million cases in the first two years of the pandemic. The estimated fatality rate lies between 1-2%, however this is considerably higher for elderly patients over 80 years of age (~10%) and nursing home residents (>20%) (1). Type I interferons impaired type I IFN response has been observed, resulting in decreased viral clearance (5). A lack of an efficient type I IFN response in these patients is in part due to inborn errors of type I IFN immunity (6) or circulating auto-antibodies neutralizing type I IFNs (7). Also, a recent animal study has identified that type I IFN signaling is required for the recruitment of pro-inflammatory cells into the lungs
To investigate the impact of impaired type I IFN signaling during SARS-CoV-2 infection, we first assessed type I IFN expression upon infection. At 2 d.p.i. both IFN-α and IFN-β were significantly increased in BAL fluid of hACE2-WT compared to eGFP-WT mice (Supp. Figure 1B). In the hACE2-Ifnar1 -/group, expression of IFN-α was significantly lower, while IFN-β levels were higher compared to the hACE2-WT group. We next investigated ISG expression after SARS-CoV-2 infection, since in Ifnar1 -/mice limited amounts of type I IFN cytokines can be produced but cannot signal for downstream ISGs induction. Chemokine Cxcl10 and anti-viral effectors Mx1, Oas1 and Viperin were quantified. The expression of these ISGs was increased in hACE2-WT mice upon infection with SARS-CoV-2 at 2 d.p.i., however in hACE2-Ifnar1 -/mice, ISG expression was significantly reduced, but not completely absent ( Figure 1F and Supp. Figure 3 C-F). These results suggest an initial increase of IFN-β in hACE2-Ifnar1 -/mice, which may be due to higher viral titers, but that is not translated into ISG expression due to the F o r P e e r R e v i e w 5 lack of signaling through the IFNAR1. We therefore investigated the expression of type III IFNs, IFN-λ2-3, which can contribute to ISG expression. IFN-λ expression was induced upon infection in hACE2-WT mice at 2 d.p.i. while remained at baseline levels in the hACE2-Ifnar1 -/mice (Supp. Figure 1C). Furthermore, Ifng gene expression was significantly increased in hACE2-Ifnar1 -/mice later during the infection, by 4 d.p.i. (Supp. Figure 4A). This correlated with CD3 + T cell recruitment to the airways (Supp. Figure 4B-D). Our data suggest that limited levels of type I or III IFNs are produced early during infection in the Ifnar1 -/mice resulting in some ISG expression but overall, these data suggest that type I IFN signaling is the main driver for inducing cell intrinsic anti-viral responses.
We next assessed the gene expression of inflammatory mediators and found that expression of the chemokine Cxcl1, which is not dependent on type I IFN signaling (3), was increased in hACE2-Ifnar1 -/mice at 2 and 4 d.p.i. compared to hACE2-WT (Figure 2A and Supp. Figure 3G). As CXCL1 plays an essential role in early host immune responses by recruiting neutrophils (11), we next analysed infiltration of neutrophils (gated as live, CD45 + , Ly6G + , Supp. Figure 5A) into the airways at 2 d.p.i. In line with highly increased gene expression of Cxcl1, neutrophil recruitment to the airways (BAL) was significantly increased in hACE2-Ifnar1 -/mice at 2 d.p.i., both proportional of leukocytes (CD45 + cells) and in total numbers ( Figure 2B and C). This was recapitulated in lung tissue with increased proportions of neutrophils in type I IFN signaling-impaired mice at 2 d.p.i. (Figure 2D and Supp. Figure   3H), decreasing over time. Taken together, these findings suggest that during SARS-CoV-2 infection, type I IFN signaling deficiency results in increased neutrophil recruitment via CXCL1, thereby contributing to a pro-inflammatory environment. Indeed, Cxcl1 is also increased in Ifnar1 -/mice during influenza A with secondary pneumococcal infection (12), but decreased during RSV infection in mice (13), highlighting a pathogen specific CXCL1 response. Furthermore, since we show similar trends for viral load and neutrophil recruitment upon SARS-CoV-2 infection (both significantly increased in hACE2-Ifnar1 -/mice), which is a mechanism present in other respiratory viral infections such as respiratory syncytial virus (RSV) (13,14), it will be important to further investigate the link between neutrophil recruitment and viral load in this model.
Since monocyte recruitment to the airways and lungs is key to early host responses to viral infection, we next investigated the expression of monocyte recruiting chemokine CCL2 and the recruitment of inflammatory myeloid cells. CCL2 protein expression was increased in BAL fluid of hACE2-WT mice at 2 d.p.i. with SARS-CoV-2 (Supp. Figure 3I). However, in hACE2-Ifnar1 -/mice CCL2 expression was significantly lower at 2 d.p.i., peaking at 4 d.p.i. at lower levels than in IFNAR1-sufficient mice ( Figure   3A). These findings are in line with a report identifying early CCR2 signaling essential to restrict viral  (15). The recruitment of CD64 + CD11b + inflammatory myeloid cells to the lung followed similar kinetics, as in hACE2-WT mice proportions were highest at 2 d.p.i. and subsequently decreased, while in IFNAR1-deficient mice proportions and total numbers of CD64 + CD11b + inflammatory myeloid cells strongly increased between 2 and 4 d.p.i. and were highest at 8 d.p.i. (Figure 3B and Supp. Figure 3J). We next assessed expression of the monocyte/macrophage differentiation antigen Ly6C within this population, since previous studies reported the infiltration of CD64 + CD11b + Ly6C + inflammatory myeloid cells into the lung during SARS-CoV-2 infection (8,15). This showed highly increased proportions of CD64 + CD11b + Ly6C + in hACE2-WT but not IFNAR1-deficient mice at 2 d.p.i. in the BAL (Supp. Figure 5D) and lung ( Figure 3C-D and Supp. Figure 3K), suggesting type I IFN dependency for recruitment. However, as we have previously shown that Ly6C is gradually downregulated on monocytes during response to respiratory viral infection (16), we also analyzed CD64 + CD11b + Ly6Ccells. The presence of CD64 + CD11b + Ly6Cinflammatory myeloid cells in the airways was not type I IFN signaling dependent, since both proportions and total numbers were significantly increased in hACE2-Ifnar1 -/mice at 4 and 8 d.p.i. (Figure 3C and E), while at 2 d.p.i.
in the airways no significant differences emerged (Supp. Figure 5F). This accounts for the delayed emergence of inflammatory myeloid cells in the lung during type I IFN signaling impairment shown in Figure 3B and overall indicates altered recruitment dynamics of inflammatory myeloid cells. Taking these data together, our model recapitulates the deficiency of type I interferon responses seen in severe SARS-CoV-2 infection, which in patients is marked by decreased IFN-α, type I IFN activity and ISG score, as well as neutrophilia and increased CCL2 (5). Our data suggest that the lack of type I IFN signaling results in dysregulated innate immune responses in the lung during SAR-CoV-2 infection.

Concluding Remarks
In summary, using a mouse model of SARS-CoV-2 infection we show that type I IFN signaling is essential for inducing anti-viral effector responses, control of virus replication and disease severity.
Our data indicate that type I IFN signaling-deficient mice express increased levels of Cxcl1 in the lung and increased infiltration of neutrophils to the airways compared to WT controls. Furthermore, we found reduced and delayed production of CCL2 and altered recruitment of inflammatory myeloid cells during IFNAR1-deficiency. This, together with an increased viral burden is associated with more severe disease in type I IFN signaling-deficient mice. The data shown here will be valuable for better understanding how impaired type I IFN signaling drives SARS-CoV-2 pathology and disease severity, which is highly relevant considering the large contribution of impaired type I IFN responses on lifethreatening SARS-CoV-2 infections (6, 7) and deaths (17) and for the development of type I IFN-based treatment options for COVID-19 in vulnerable populations. To conclude, our findings show that type I

Materials and Methods
Mice C57BL/6 mice were purchased from Charles River UK Inc. Ifnar1 -/mice on a C57BL/6 background were bred in-house. All mice were bred and maintained in pathogen-free conditions and 8-12-week-old mice were used for experiments. All animal experiments were reviewed and approved by the Animal Welfare and Ethical Review Board (AWERB) at Imperial College London and approved by the UK Home Office in accordance with the Animals Act 1986 (Scientific Procedures) and ARRIVE guidelines. Both male and female mice were used for experiments after excluding sex bias in preliminary experiments.
All experiments were performed twice, independently, per time point.
Purity of vectors was confirmed by analyzing 20 μl of diluted vector on 4-12% SDS polyacrylamide gels, where total protein was visualized using Coomassie stain according to the manufacturer's protocols (Life Technologies).

Cryosectioning and native eGFP detection
Mice were sacrificed 20 days post instillation of AAV-eGFP or PBS and lungs were removed after inflation with 4% PFA. After 24-hour fixation in 4% PFA, lungs were inflated with 30% sucrose and submerged in 30% sucrose for 24 hours. Lungs were subsequently inflated with 1:1 cryo embedding matrix (OCT)/30% sucrose and individual lobes were submerged in OCT/30% sucrose in plastic molds and frozen at -80 °C. Left lungs were cryosectioned to produce 7 μm thick sections, mounted using DAPI-supplemented mounting media with coverslip, and eGFP expression was detected by fluorescent microscopy using the EVOS FL Auto 2 system (Thermo Scientific).

Virus and infections
First wave SARS-CoV-2 (D614G, isolate of hCoV-19/England/IC19/2020) was grown in African green monkey kidney cells overexpressing human ACE2 and TMPRSS2 (Vero-ACE2-TMPRSS2; VAT cells) (22). h at 37°C. The inoculum was then removed and replaced with overlay medium (1x MEM, 0.2% w/v BSA, 0.16% w/v NaHCO 3 , 10 mM HEPES, 2 mM L-Glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.84% agarose). Plates were incubated for 3 days at 37°C before overlay was removed and cells were stained for 1 h at room temperature in 2x crystal violet solution. Virus plaques were counted and multiplied by the dilution factor to calculate titer as PFU/ml.

Flow cytometry
After red blood cell lysis, lung and BAL cells were incubated for 30 min with fixable live-dead Aqua dye (Invitrogen), followed by fixation for 30 minutes with 4% paraformaldehyde (PFA) to inactivate virus.
Cells were then incubated for 20 min with a purified rat IgG 2b anti-mouse CD16/CD32 receptor antibody (BD) to block

RNA isolation and quantitative RT-PCR
Lung tissue was homogenized in TRIzol and RNA extraction performed according to manufacturer's instructions. After the chloroform step, the aqueous phase containing RNA was further processed using the RNeasy Mini Kit (QIAGEN) according to manufacturer's instructions. 2 µg RNA was reverse transcribed using a High-Capacity RNA-to-cDNA kit (Applied Biosystems) according to manufacturer's instructions. To quantify mRNA levels in lung tissue, quantitative RT-PCR reactions for Oas1, Viperin and Ifnl were performed using primers and probes as previously described (23). Analysis was performed using the QuantiTect Probe PCR Master Mix (QIAGEN) and the 7500 Fast real-Time PCR System (Applied Biosystems). For absolute quantification, the exact number of copies of the gene of interest was calculated using a plasmid DNA standard curve, and the results were normalized to levels of Gapdh (Applied Biosystems). For relative quantification, the expression of Cxcl1, Cxcl10, hACE2, Mx1 and SARS-CoV-2 N and E gene was expressed relatively to the expression of Gapdh. First, the ΔCT (CT = cycle threshold) between the target gene and Gapdh was calculated for each sample, followed by calculation of 2 -ΔCT . Analysis was performed using 7500 Fast System SDS Software (Applied Biosystems).  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59